Abstract
Objectives: To investigate the geographic distribution of common cystic fibrosis (CF) variants in the western and southern regions of Saudi Arabia.
Methods: A retrospective study was conducted on 69 patients diagnosed with CF at King Faisal Specialist Hospital & Research Center, Jeddah. Patient data were collected retrospectively between June 2000 and November 2021. Various parameters were considered, including patient demographic information, CFTR variants, and respiratory cultures.
Results: We identified 26 CFTR variants in 69 patients with CF, including one novel variant that had not been reported or published before (1549del G) in 2 patients with CF. The 6 most prevalentvariants were as follows: c.1521_1523delCTT (19%), c.1418delG (10.2%), c.579+1G>T (8.8%), c.2988+1G>A (8.8%), c.3419 T>A (7.2%), and c.4124A>C (5.8%). In addition, respiratory cultures revealed that Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Streptococcus pneumoniae were highly common among patients with CF.
Conclusion: This study highlighted features of patients with CF residing in the Western and Southern regions of Saudi Arabia. Six of the 26 CFTR variants were common in these patients. We also report, for the first time, a novel variant and other CFTR variants that are yet to be reported in Saudi Arabia. These findings could help establish a foundation for cystic fibrosis screening in Saudi Arabia and may assist in clinical diagnosis and prognosis.
Cystic fibrosis (CF) is an autosomal recessive disease due to a mutation of the gene on human chromosome 7 that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) protein.1 The primary function of CFTR is to create chloride channels for mucus transport via chloride secretion.2 The CFTR gene mutations can affect chloride release, sodium reabsorption, and water transport.1,3 This disruption leads to dehydrated mucus secretion and an inflammatory response, contributing to severe bronchiectasis and, ultimately, respiratory failure.4 Imaging techniques including chest radiography, magnetic resonance imaging, and computed tomography, are commonly used to diagnose CF.5,6
More than 2000 variants of CFTR gene have been described in the Cystic Fibrosis Mutation Database.7 In addition, there are 6 different classes of CFTR gene mutations depending on the defects. Class I is characterized by a deficiency in CTFR protein production, whereas class II arises from abnormalities in post-translational processing.8,9 Classes III, IV, V, and VI arise from defects in channel activity, reductions in ion flow rate and duration, functional CFTR activity, and CFTR stability.8-10
The incidence of CF in the Middle East is 1 in 2500-5000, and one in 4243 in Saudi Arabia.11,12 Deletion of phenylalanine at position 508 (F508del) is the most common variant detected in CF, specifically in the Caucasian population.8,13 Other variants, including G542X, W1282X, and N1303K, have been found in Europe, North Africa, and Latin America.10,14 Between 1998 and 2017, 312 families were positive for CFTR variants, and consanguinity was observed in 85% of the parents.15
In this study, we provide an overview of the characteristics of patients with CF and the spreading of CFTR mutations among Saudi citizens in the western and southern regions of Saudi Arabia. This investigation will help identify the most common CFTR variants and understand the colonialization of bacteria in patients with CF, which could support clinical screening and prognosis.
Methods
This retrospective study utilized the medical records of patients with CF between June 2000 and November 2021 at King Faisal Specialist Hospital & Research Centre in Jeddah, Saudi Arabia. Data from patients residing in the western (Makkah, Jeddah, and Tabuk provinces) and southern regions (Baha, Jizan, Asir, and Najran provinces) who visited the hospital were incorporated in our study. To investigate the CFTR variants in the 69 patients, we used 3 CF mutation databases: Cystic Fibrosis Mutation Database, The Human Gene Mutation Database, and Clinical and Functional Translation of CFTR (CFTR2).
Data were recruited for analysis after the incidence of CF was confirmed by: i) Assessing the standard clinical image of sputum, cough production, and family history of CF. ii) High sweat chloride levels (>60mmol/L) in 2 consequent samples using the Wescor quantitative test. iii) Genetic detection of CFTR variants by next generation sequencing. Demographic information, such as gender, geographic location, and age of patients with CF, was initially included in the analysis.
This study was approved by the ethics committee International Review Board in King Faisal Specialist Hospital & Research Center, Jeddah, (IRB#: 2023-41) following the Declaration of Helsinki and good clinical practice guidelines. All collected data were reviewed and supervised by the principal investigator.
Statistical analysis
Descriptive analyses were completed to calculate the distribution patterns of parameters in the sample, such as gender, age, the geographic distribution of CF patients, common CFTR variants, and bacterial detection in percentages.
Results
Of the 69 diagnosed patients with CF, 40 (58%) were male and 29 (24%) were female. Patients with CF were categorized into 6 groups based on age: <1, 1–5, 6–10, 11–15, 16–20, and >20 years old (Table 1). In addition, 89.9% of the patients with CF in the King Faisal Specialist Hospital & Research Centre were residents of the western region, whereas 10.1% were residents of the southern region of Saudi Arabia.
- The age distribution of patients with CF featured in this study.
The identification of CFTR variants
This study identified 26 CFTR variants in 69 patients with CF (Table 2). The variants have been described in the CFTR databases and documented in the literature. However, we identified a novel variant that has not been reported or published in the literature. In addition, the prevalence of the variants was variable, as some appeared in more than 5 patients, while others appeared only once. For example, the c.220C>T and c.530T>C variants were observed in more than one patients.
- Cystic fibrosis transmembrane conductance regulator (CFTR) variants identified in patients with cystic fibrosis from Saudi Arabia. The identity of the complementary deoxyribonucleic acid (cDNA), protein, legacy, and exon region of each identified CFTR variant is listed.
Among the 26 CFTR variants observed in this study, 6 common variants were detected in 69 patients with CF. The most common variants identified were as follows: i) c.1521_1523delCTT (19%), ii) c.1418delG (10.2%), iii) c.579+1G>T (8.8%), iv) c.2988+1G>A (8.8%), v) c.3419 T>A (5.8%), and vi) c.4124A>C (5.8%), as shown in Table 3.
- The number of patients retaining common cystic fibrosis transmembrane conductance regulator variants and their genotype.
We also found 19 variants in 34 patients with CF residing in Jeddah, where the most prevalent variant was c.1521_1523delCTT (26.3%). In contrast, 12 variants were identified in 14 patients with CF from Makkah; the most common variant was c.3419 T>A (33%).
Interestingly, 3 patients harbored more than one variant. The first patient was homozygous for 3 variants: c.1408G>A, c.2562T>G, and c.4389G>A. The second patient was heterozygous for 3 variants (c.869+11C>T, c.1408G>A, and c.91C>T), and the third was homozygous for 2 variants (c.1399C>T and c.1521_1523delCTT).
Notably, a novel CFTR variant was identified in 2 patients (a female and a male). Both patients were from the Western region. However, their ages varied, where the females were 5 and males were 11 years old. In addition, the CFTR trait in both patients was homozygous. The respiratory cultures for the female patient revealed Staphylococcus aureus (S. aureus) while the culture for the male patient showed Streptococcus pneumoniae (S. pneumonia).
Respiratory cell culture
Respiratory culture data from 54 patients with CF revealed the presence of several bacterial species (Appendix 1). The predominant species observed in most of the samples were Pseudomonas aeruginosa (P. aeruginosa) (44.4%), Haemophilus influenzae (H. influenzae) (18.5%), S. aureus (18.5%), S,pneumoniae (13%), and Branhamella catarrhalis (B. catarrhalis) (9%). Although P. aeruginosa was the most common bacterial type detected in all patients, its prevalence was lower in females (12.9%) than in males (31.5%). We also found that these species were all associated with the 4 most common CFTR variants: c.1521_1523delCTT, c.1418delG, c.579+1G>T, and c.2988+1G>A. Other bacterial species, such as Klebsiella sp. (3%) and Stenotrophomonas maltophilia (S. maltophilia) (1.8%), were found only rarely (Table 4).
- Types of bacterial colonization from the first respiratory culture in patients with CF with common CFTR variants.
Discussion
Cystic fibrosis has attracted the attention of researchers attempting to understand and explore the incidence and genetic mutations in the CFTR gene. Likewise, this study aimed to investigate the genetic variation in CFTR genes in Saudi patients with CF. However, we focused mainly on the western and southern regions of Saudi Arabia. Various parameters were considered, such as demographic information, CFTR gene variant identity, and bacterial colonization associated with the gene variants. Since the primary goal of this study was to understand the characteristics of Saudi patients diagnosed with CF in the western and southern regions only, 69 CF cases were identified between 2000 and 2021. This explains why the sample size of this study was comparatively small compared to another study (396 patients with CF) that targeted the general population.15 Several studies have suggested that gender can predict the clinical outcomes and severity of CF. It has been demonstrated that females risk a worse prognosis than males because of increased respiratory infections, and P. aeruginosa has been implicated in poor survival.41,42 This suggests that female patients with CF from the western and southern regions presumably have a better survival rate since the number of female patients and the incidence of P. aeruginosa were low.
Banjar et al43 provided the first description of CFTR gene variants in the Saudi population in 1999. Several studies by the Banjar group have explored CF variants in Saudi Arabia over various time frames.12,15,17 The high incidence of homozygous CF cases observed in this study (81%) reflected the occurrence of consanguineous marriages. This has also been reported in other studies, where consanguinity accounted for 89% of the families with CF.44
The c.1521_1523delCTT variant, responsible for the deletion of phenylalanine at position 508 in the CFTR gene (p.Phe508del), is considered the most common variant in CF.45 This is consistent with the findings of this study, as 19% of CF cases had p.Phe508del. It has also recently been reported that 70% of patients with CF in the western region of Saudi Arabia exhibit this mutation.17,43,46 According to the findings of our study, the c.1418delG variant was found to be most common in the northern region. The third most common variant, c.579+1G>T, was discovered to be more common in the southern region. Lastly, c.2988+1G>A, the fourth common variant identified in our study, was most frequently found in the eastern region.17 Interestingly, c.3419T>A (p.Met1140Lys), which accounted for 7.2% of the cases in this study, has been reported in only one patient in Saudi Arabia, and c.4124A>C (p.His1375Pro) (in 5.8% of our cases) has not been previously reported in Saudi patients with CF.47
In addition, our study reports variants of the CFTR gene that have not been previously reported in the Saudi population. However, these variants have been reported or published in the literature in other Arab and American populations.46,48 The variants are c.443T>C, c.2657+5G>A, c.1697C>A, c.2620-26A>G, c.2051_2052delAAinsG, c.1660_1661insA, c.4251delA, c.3717+12191C>T, c.220C>T, c.530T>C, c.1408A>G, c.2562T>G, c.4389G>A, c.869+11C>T, c.91C>T and c.1399C>T. The clinical significance of these variants differs; they are considered to be either pathogenic, likely pathogenic, or benign, or there are conflicting interpretations of pathogenicity (Appendix 1).23-30,32-38,40 Further studies are essential to clarify the clinical significance of these variants. We also found a CFTR variant (delExone 19-21) present in 2 patients with CF, which has been reported previously in the Saudi population.15
Interestingly, we identified a novel variant of the CFTR gene. The variant is (1549delG), located on exon 10. It is predicted to cause deletion of a “G” at nucleotide position 1549 in the CFTR gene and subsequently causes a frameshift mutation which alters the sequence of the amino acid.
Pathogenic bacteria are among the primary factors contributing to morbidity and mortality in CF.49 Pseudomonas aeruginosa, S. aureus, and H. influenzae are among the most prevalent pulmonary bacterial infections associated with CF.50,51 In addition, infections caused by multiple species have been identified. These include co-infections with bacteria and fungi.52 Although culture-based studies are the most common method used to detect and assess infection in patients with CF, further studies are recommended to explore the role of these microorganisms in pathogenesis.53,54 In this study, most patients who underwent respiratory culture examination displayed colonization by P. aeruginosa, S. aureus, H. influenzae, S. pneumoniae, and B. catarrhalis. This is consistent with the most common CF lung bacterial species.54,55 These species were also associated with the 4 most common CFTR variants (c.1521_1523delCTT, c.1418delG, c.579+1G>T, and c.2988+1G>A) and with other less common variants. Notably, no correlation was found between the type of mutation and bacterial species, corresponding to Banjar et al’s56 findings.
Some bacterial species, such as S. maltophilia and Klebsiella pneumoniae (Klebsiella sp.), were detected infrequently in this study. Several studies have suggested that S. maltophilia is less pathogenic than other species and is not related with the lung diseases.57-59 Another study suggested that Klebsiella sp. is rarely found in patients with CF, which reflects what we found in Saudi patients with CF. 60
Study limitations
Since the diagnosis within a medical record is a prevalent theme, health care institutions should be provided with adequate training and facilities to enable them to make accurate early diagnoses and, in turn, appropriate treatments with high cure rates. Data from medical reports and registries that have been analyzed have shown considerable limitations in the reporting of standardized information on cystic fibrosis from health care settings. The implementation of medical staff training and infrastructure is crucial to sustaining this concern and bringing forth clear conclusions. One of the limitations of our study was the presence of incomplete medical records or patients who did not follow up after receiving care.
To verify our findings, a larger multicenter cohort investigation is required. Further, the majority of errors in sweat chloride tests are caused by insufficient sweat collection, technological problems, and misinterpretation of data. The apparent low incidence of diagnosed CF patients in Saudi Arabia could be owing to a lack of clinical suspicion, as well as technical errors in sweat chloride level detection and a lack of autopsy data. A higher level of suspicion is required for an early diagnosis. A comprehensive national program would be advantageous in raising awareness among general physicians and pediatricians in Saudi Arabia about the occurrence and potential devastating consequences of CF.
In conclusion, this study highlighted the characteristics of patients with CF in the western and (to a lesser extent) southern regions of Saudi Arabia. Multiple CFTR variants were found in patients with CF, but only 6 were common. Here, we report for the first time a novel CFTR variant (1549del G). In addition, the majority of patients representing these regions were infected with P. aeruginosa, S. aureus, H. influenzae, and S. pneumoniae. These findings could help assess clinical prognosis and predict life expectancy, outcomes, and possible treatment strategies. However, a patient’s follow-up with these clinical features will assist in measuring the pathological consequences of CF on health.
Acknowledgment
Yousef Hawsawi and Rimah Saleem would like to thank Alfaisal University, Riyadh, Kingdom of Saudi Arabia for their support. We also would like to thank Editage (www.editage.com) for the English language editing.
Appendix
- A description of the consequence, clinical significance, genetic variation and condition of the 26 identified CFTR variants.
Footnotes
Disclosure. Authors have no conflict of interests, and the work was not supported or funded by any drug company.
- Received July 31, 2023.
- Accepted August 31, 2023.
- Copyright: © Saudi Medical Journal
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